Functioning of the electromechanical connection in the course of the contracture contraction

The effects of calcium release blocker dantrolene was tested on electrically evoked twitches and on contractures induced by potassium depolarization, by acetylcholine or caffeine. It was shown that the first: developmental, stage of potassium or acetylcholine contracture is inhibited by dantrolene and is not influenced by calcium free medium, therefore we may interpret it as based on a "voltage-dependent Ca release" (VDCR) mechanism of activation, whereas depolarization directly opens the rhyanodin receptor calcium channels. On the contrary, the next stage: the long-lasting plateau of contracture, is directly dependent on external Ca2+ and inhibited by dantrolene, and therefore can be described as "calcium induced Ca-release" (CICR) activation mechanism. In this case stored calcium is also released by rhyanodine receptors, although by means of entering the extracellular Ca2+. Finally, the last stage of low amplitude is not influenced by dantrolene nor by calcium-free medium. Therefore the activation of contraction on this stage is not based on the Ca2+ release through the rhyanodin receptor calcium channels.     

Effects of calcium antagonists on contraction of a holothurian muscle

Contractions evoked by acetylcholine or by tetanic stimulation differed from caffeine contractures, in muscle strips isolated from the longitudinal muscle bands of the body wall of Isostichopus badionotus (Selenka), an aspidochirote holothurian. Tetanic contractions and responses to acetylcholine remained reproducible for hours in sea water or artificial sea water. Caffeine contractures declined rapidly during a series of repetitions, carried out in a bath medium which had a calcium content equivalent to that of sea water. Manganese, lathanum, and dantrolene have been used as calcium antagonists, with the objective of differentiating the calcium stores used in reproducible contractions from the calcium stores used in rapidly extinguishing contractures. Muscle strips were pretreated with an ionophore (X-537A) to confer reproducibility in a series of caffeine contractures, before use of calcium antagonists. For reproducible caffeine contractures, the order of effectiveness of calcium antagonists was lanthanum greater than manganese greater than dantrolene. The order of reversibility was manganese greater than dantrolene greater than lanthanum. For acetylcholine contractions, or tetanic contractions, the order of effectiveness of calcium antagonists was lanthanum greater than manganese and the order of reversibility was manganese greater than lanthanum. Dantrolene reversibility enhanced contractions.     

Molecular Mechanisms of Regulation of the Activity of Sarcoplasmic Reticulum Ca-Release Channels (Ryanodine Receptors), Muscle Fatigue, and Severin''s Phenomenon

In this short review of the literature and our own data the characteristics of structural organization of sarcoplasmic reticulum Ca-release channels (ryanodine receptors) in different types of muscles, the participation of other sarcoplasmic reticulum proteins in excitation–contraction coupling and Ca-release channel operation, and the regulation of the channel activity by endogenous low molecular weight compounds are analyzed. Special attention is given to changes that occur in muscle cells during exhausting work and to the role of sarcoplasmic reticulum Ca-release channels in the loss of muscle contractile activity during the development of fatigue. It is concluded that the protection of muscle fibers against fatigue in the presence of the histidine-containing dipeptide carnosine, called in the literature Severin's phenomenon, is primarily connected with modulation of sarcoplasmic reticulum Ca-release channel activity by carnosine.     

The regulation of muscle phosphorylase kinase by calcium ions,calmodulin and troponin-C

Although it has been believed for several years that calcium ions are the means by which glycogenolysis and muscle contraction are synchronized, it is only over the past three years that this concept has started to be placed on a firm molecular basis. It appears that the regulation of phosphorylase kinase $\text{in vivo}$ is achieved through the interaction of the enzyme with the two calcium binding proteins, calmodulin and troponin-C, and that the relative importance of these proteins depends on the degree of phosphorylation of the enzyme (figure 3). In the dephosphorylated form of the enzyme, troponin-C rather than calmodulin is the dominant calcium dependent regulator providing an attractive mechanism for coupling glycogenolysis and muscle contraction, since the same calcium binding protein activates both processes. On the other hand, the phosphorylated form of the enzyme can hardly be activated at all by troponin-C, although it is still completely dependent on calcium ions. Calmodulin (the δ - subunit) is therefore the dominant calcium dependent regulator of phosphorylase kinase in its hormonally activated state.

Recent work has demonstrated that phosphorylase kinase not only activates phosphorylase, but also phosphorylates glycogen synthase thereby decreasing its activity (45–49). The regulation of phosphorylase kinase by calcium ions may therefore also provide a mechanism for co-ordinating the rates of glycogenolysis and glycogen synthesis during muscle contraction.     

Effects of modulators of sarcoplasmic Ca2+ release on the development of skeletal muscle fatigue.

The reduced release of Ca2+ from sarcoplasmic reticulum (SR) is considered a major determinant of muscle fatigue. In the present study, we investigated whether the presence of dantrolene, an established inhibitor of SR Ca2+ release, or caffeine, a drug facilitating SR Ca2+ release, modifies muscle fatigue development. Accordingly, the effects of Ca2+ release modulators were analyzed in vitro in mouse fast-twitch [extensor digitorum longus (EDL)] and slow-twitch (soleus) muscles, fatigued by repeated short tetani (40 Hz for 300 ms, 0.5 s(-1) in soleus and 60 Hz for 300 ms, 0.3 s(-1) in EDL, for 6 min). Caffeine produced a substantial increase of tetanic tension of both EDL and soleus muscles, whereas dantrolene decreased tetanic tension only in EDL muscle. In both EDL and soleus muscles, 5 microM dantrolene did not affect fatigue development, whereas 20 microM dantrolene produced a positive staircase during the first 3 min of stimulation in EDL muscle and a slowing of fatigue development in soleus muscle. The development of the positive staircase was abolished by the addition of 15 microM ML-7, a selective inhibitor of myosin light chain kinase. On the other hand, caffeine caused a larger and faster loss of tension in both EDL and soleus muscles. The results seem to indicate that the changes in fatigue profile induced by caffeine or dantrolene are mainly due to the changes in the initial tetanic tension caused by the drugs, with the resulting changes in the level of contraction-dependent factors of fatigue, rather than to changes in the SR Ca2+ release during fatigue development.     

Characteristics of functioning of electromechanical coupling in striated muscles of higher and lower vertebrates

A comparative pharmacological analysis of relative contributions of different signal transduction pathways in the activation of contraction (excitation-contraction coupling, ECC) in intact fast striated muscles of frog and lamprey was performed. It was found that the major mechanism responsible for the ECC in muscles of both animals is Ca2+ release from the sarcoplasmic reticulum through the ryanodine-sensitive channels. However, the ECC in lamprey muscle displays some important differences in the units of electromechanical coupling, which precede the calcium release from sarcoplasmic reticulum. The maximum contraction force in frog muscle develops during caffeine-induced contracture, which indicates that all Ca2+ stored in sarcoplasmic reticulum is released through ryanodine-sensitive channels. In contrast, in lamprey muscle, the maximum force develops not in response to high caffeine concentration, but in response to repetitive electrical stimulation. Hence, in addition to stores liberated by ryanodine-sensitive channels, some other sources of calcium ions should exist, which contribute to the contraction activation. A source of this additional Ca2+ ions can be external medium, because acetylcholine contracture is abolished in a calcium-free medium. In frog muscle, the acetylcholine contracture was abolished in a Na(+)-free solution. It was concluded that in frog muscle ECC can be triggered by changes in the transmembrane potential (depolarization-induced calcium release), while in lamprey muscle the entry of calcium ions into myoplasm as the trigger in ECC (calcium-induced calcium release). The lamprey muscle was found to be more resistant to tetrodotoxin and tetracaine, which is indicative of a role in the activation of contraction of tetrodotoxin-resistant Na+ and/or Ca2+ channels. It was concluded, that ECC mechanism in striated muscles of low vertebrates is not limited by the generally accepted scheme of depolarization-induced calcium release but can include some other schemes, which require the Ca2+ influx into the cell.     

Different effects of verapamil and low calcium on repetitive contractile activity of frog fatigue-resistant and easily-fatigued muscle fibres.

The effects of low calcium and verapamil on contractility of two muscle fibre types (m. iliofibularis, Rana temporaria) upon different stimulation protocols were been compared. Verapamil (0.02 mmol/l) induced temporal excitation-contraction coupling failure during single tetanic stimulation and enhanced the decline of tetanic force during 30 s repetitive tetanic stimulation in both fatigue-resistant fibres and easily-fatigued fibres. In contrast to verapamil, low extracellular calcium (0.02 mmol/l) only enhanced the decline of tetanic force in fatigue-resistant during repetitive tetanic stimulation but had no effect on easily-fatigued fibres. The effect of verapamil on the decline of tetanic force in fatigue-resistant fibres was more profound in low calcium conditions. Both verapamil and low calcium eliminated twitch facilitation that appeared after prolonged contractile activity in fatigue-resistant fibres. 4mmol/l Ni+2, used as calcium channel antagonist, had effects similar to low calcium medium. It could be concluded that (i) extracellular Ca2+-requirements for excitation-contraction coupling are different in fatigue-resistant and easily-fatigued fibres; (ii) the effects of verapamil on force performance are not entirely dependent upon calcium channel blockade.     

Activation in a Skeletal Muscle Contraction Model with a Modification for Insect Fibrillar Muscle

A sliding filament model for muscle contraction is extended by including an activation mechanism based on the hypothesis that the binding of calcium by a regulating protein in the myofibrils must occur before the rate constant governing the making of interactions between cross-bridges and thin filament sites can take on nonzero values. The magnitude of the rate constant is proportional to the amount of bound calcium. The model's isometric twitch and rise of force in an isometric tetanus are similar to the curves produced by real muscles. It redevelops force after a quick release in an isometric tetanus faster than the initial rise. Quick release experiments on the model during an isometric twitch show that the “active state” curve produced is different from the postulated calcium binding curve. The force developed by the model can be increased by a small quick stretch delivered soon after activation to values near the maximum generated in an isometric tetanus. Following the quick stretch, the force remains near the tetanic maximum for a long time even though the calcium binding curve rises to a peak and subsequently decays by about 50%. The model satisfies the constraint of shortening with a constant velocity under a constant load. Modifications can be made in the model so that it produces the delayed force changes following step length changes characteristic of insect fibrillar muscle.     

Dantrolene stabilizes domain interactions within the ryanodine receptor

Interdomain interactions between N-terminal and central domains serving as a "domain switch" are believed to be essential to the functional regulation of the skeletal muscle ryanodine receptor-1 Ca(2+) channel. Mutational destabilization of the domain switch in malignant hyperthermia (MH), a genetic sensitivity to volatile anesthetics, causes functional instability of the channel. Dantrolene, a drug used to treat MH, binds to a region within this proposed domain switch. To explore its mechanism of action, the effect of dantrolene on MH-like channel activation by the synthetic domain peptide DP4 or anti-DP4 antibody was examined. A fluorescence probe, methylcoumarin acetate, was covalently attached to the domain switch using DP4 as a delivery vehicle. The magnitude of domain unzipping was determined from the accessibility of methylcoumarin acetate to a macromolecular fluorescence quencher. The Stern-Volmer quenching constant (K(Q)) increased with the addition of DP4 or anti-DP4 antibody. This increase was reversed by dantrolene at both 37 and 22 degrees C and was unaffected by calmodulin. [(3)H]Ryanodine binding to the sarcoplasmic reticulum and activation of sarcoplasmic reticulum Ca(2+) release, both measures of channel activation, were enhanced by DP4. These activities were inhibited by dantrolene at 37 degrees C, yet required the presence of calmodulin at 22 degrees C. These results suggest that the mechanism of action of dantrolene involves stabilization of domain-domain interactions within the domain switch, preventing domain unzipping-induced channel dysfunction. We suggest that temperature and calmodulin primarily affect the coupling between the domain switch and the downstream mechanism of regulation of Ca(2+) channel opening rather than the domain switch itself.     

Aminophylline enhances contractility of frog skeletal muscle: an effect dependent on extracellular calcium

The effects of aminophylline (10-500 microM) on isometric twitch and tetanic forces were studied in vitro on frog semitendinosus muscle. Two hypotheses were tested: 1) that micromolar concentrations of aminophylline enhanced contractility of isolated skeletal muscle and 2) that the potentiating effect of aminophylline was dependent on the presence of extracellular calcium ions. Muscles were removed, placed in aerated Ringer solution at 20 degrees C, attached to a force transducer, and stimulated directly. Muscles in normal Ringer and aminophylline Ringer were compared throughout the frequency-force relationship from twitches to maximum tetanic force. Aminophylline increased twitch force significantly at concentrations as low as 25 microM. Over a range of stimulation frequencies, but especially at 10 and 20 Hz, aminophylline increased tetanic force. The potentiating effect of aminophylline (100 microM) was reduced or eliminated in calcium-free Ringer containing 10 mM magnesium. We conclude that aminophylline, at therapeutic concentrations, enhances muscle contractility, and the enhancement is dependent on the presence of extracellular calcium. These findings support the concept that aminophylline is effective in improving respiration in humans with airway obstruction by enhancing diaphragmatic contractility.     

What drives activation-dependent shifts in the force–length curve?

Skeletal muscles are rarely recruited maximally during movement. However, much of our understanding of muscle properties is based on studies using maximal activation. The effect of activation level on skeletal muscle properties remains poorly understood. Muscle optimum length increases with decreased activation; however, the mechanism responsible is unclear. Here, we attempted to determine whether length-dependent calcium effects, or the effect of absolute force underpin this shift. Fixed-end contractions were performed in frog plantaris muscles at a range of lengths using maximal tetanic (high force, high calcium), submaximal tetanic (low force, high calcium) and twitch (low force, low calcium) stimulation conditions. Peak force and optimum length were determined in each condition. Optimum length increased with decreasing peak force, irrespective of stimulation condition. Assuming calcium concentration varied as predicted, this suggests that absolute force, rather than calcium concentration, underpins the effect of activation level on optimum length. We suggest that the effect of absolute force is due to the varying effect of the internal mechanics of the muscle at different activation levels. These findings have implications for our understanding of in vivo muscle function and suggest that mechanical interactions within muscle may be important determinants of force at lower levels of activation.     

Models of Calmodulin Trapping and CaM Kinase II Activation in a Dendritic Spine

Activation of calcium/calmodulin-dependent protein kinase II (CaMKII) by calmodulin following calcium entry into the cell is important for long-term potentiation (LTP). Here a model of calmodulin binding and trapping by CaMKII in a dendritic spine was used to estimate levels and durations of CaMKII activation following LTP-inducing tetani. The calcium signal was calcium influx through NMDA receptor channels computed in a highly detailed dentate granule cell model. Calcium could bind to calmodulin and calmodulin to CaMKII. CaMKII subunits were either free, bound with calmodulin, trapped, autonomous, or capped. Strong low-frequency tetanic input produced little calmodulin trapping or CaMKII activation. Strong high-frequency tetanic input caused large numbers of CaMKII subunits to become trapped, and CaMKII was strongly activated. Calmodulin trapping and CaMKII activation were highly dependent on tetanus frequency (particularly between 10 and 100 Hz) and were highly sensitive to relatively small changes in the calcium signal. Repetition of a short high-frequency tetanus was necessary to achieve high levels of CaMKII activation. Three stages of CaMKII activation were found in the model: a short, highly activated stage; an intermediate, moderately active stage; and a long-lasting third stage, whose duration depended on dephosphorylation rates but whose decay rate was faster at low CaMKII activation levels than at high levels. It is not clear which of these three stages is most important for LTP.     

The effects of pH on the kinetics of fatigue and recovery in frog sartorius muscle

The effects of pH on the kinetics of fatigue and recovery in frog sartorius muscle were studied to establish whether the pH to which muscles are exposed (extracellular pH) has an effect on both the rate of fatigue development and recovery from fatigue. When frog sartorius muscles were stimulated with short tetanic stimuli at rates varying from 0.2 to 2.0 trains/s, a time- and frequency-dependent decrease in force development was observed, but extracellular pH had comparatively little effect. The recovery of tetanic force was dependent on the extracellular pH. This effect was characterized by a rapid recovery in force at pH 8.0 and an inhibition of recovery at pH 6.4 even when force decreased by only 25% during stimulation. Even when muscles were fatigued at pH 8.0 the rate of force recovery was still very small at pH 6.4. A model is proposed in which a step of the contraction cycle changes from a normal to a fatigued state. The rate of this transition is a function of the stimulation frequency and not pH. The reverse transition, from a fatigued to normal state is pH dependent; i.e., it is inhibited by H+. Measurements of resting and action potentials show that extracellular pH influences these parameters in the fatigue state, but there is no evidence that these changes are directly responsible for the pH-dependent step in the reversal of fatigue.     

Cholinesterases and choline acetyltransferase in the longitudinal muscle of the guinea pig ileum

In order to gain insight into the possible role of the ACh-system in the smooth muscle cell, the presence of choline acetyltransferase, acetylcholinesterase and butyrylcholinesterase was studied in the longitudinal muscle of the guinea-pig ileum after the mechanical removal of Auerbach's plexus. Such treatment completely removes all nerve elements as confirmed by histochemistry and electron-microscopic examination. It was found that in the longitudinal muscle devoid of all nervous elements a substantial percentage of the activity of all three enzymes still remained. Ultrastructural localization of acetylcholinesterase and butyrylcholinesterase was observed on the sarcolemma, sarcoplastic reticulum, nuclear membrane and invaginations of the sarcolemma. The localization of cholinesterases coincides with sites which are presumably involved in calcium movements during contraction and relaxation. It is well known that the depolarized smooth muscle responds to exogenous ACh with a reversible, calcium dependent contraction and it was suggested that ACh may act by increasing the influx of calcium through the cell membrane or by liberating calcium from its bound form. The presence of choline acetyltransferase and cholinesterase activities in the muscle cell proper, as well as the localization of cholinesterases on structures connected with calcium movements, support the coexistence of an intrinsic cholinergic mechanism in the smooth muscle.     

Skeletal muscle contractile performance and ADP accumulation in adenylate kinase-deficient mice

The production of AMP by adenylate kinase (AK) and subsequent deamination by AMP deaminase limits ADP accumulation during conditions of high-energy demand in skeletal muscle. The goal of this study was to investigate the consequences of AK deficiency (–/–) on adenine nucleotide management and whole muscle function at high-energy demands. To do this, we examined isometric tetanic contractile performance of the gastrocnemius-plantaris-soleus (GPS) muscle group in situ in AK1^–/– mice and wild-type (WT) controls over a range of contraction frequencies (30–120 tetani/min). We found that AK1^–/– muscle exhibited a diminished inosine 5'-monophosphate formation rate (14% of WT) and an inordinate accumulation of ADP (1.5 mM) at the highest energy demands, compared with WT controls. AK-deficient muscle exhibited similar initial contractile performance (521 ± 9 and 521 ± 10 g tension in WT and AK1^–/– muscle, respectively), followed by a significant slowing of relaxation kinetics at the highest energy demands relative to WT controls. This is consistent with a depressed capacity to sequester calcium in the presence of high ADP. However, the overall pattern of fatigue in AK1^–/– mice was similar to WT control muscle. Our findings directly demonstrate the importance of AMP formation and subsequent deamination in limiting ADP accumulation. Whole muscle contractile performance was, however, remarkably tolerant of ADP accumulation markedly in excess of what normally occurs in skeletal muscle. AMP deaminase; tetanic contraction; muscle relaxation; calcium handling; cross-bridge cycling     

Caffeine potentiates low frequency skeletal muscle force in habitual and nonhabitual caffeine consumers.

The mechanism of action underlying the ergogenic effect of caffeine is still unclear. Caffeine increases the force of muscular contraction during low-frequency stimulation by potentiating calcium release from the sarcoplasmic reticulum. Studies have also suggested an enhancement of lipid oxidation and glycogen sparing as potential mechanisms. Given that several studies have found an ergogenic effect of caffeine with no apparent metabolic effects, it is likely that a direct effect upon muscle is important. Twelve healthy male subjects were classified as habitual (n = 6) or nonhabitual (n = 6) caffeine consumers based on a 4-day diet record analysis, with a mean caffeine consumption of 771 and 14 mg/day for each group, respectively. Subjects were randomly allocated to receive caffeine (6 mg/kg) and placebo (citrate) in a double-blind, cross-over fashion approximately 100 min before a 2-min tetanic stimulation of the common peroneal nerve in a custom-made dynamometer (2 trials each of 20 and 40 Hz). Tetanic torque was measured every 30 s during and at 1, 5, and 15 min after the stimulation protocol. Maximal voluntary contraction strength and peak twitch torque were measured before and after the stimulation protocol. Caffeine potentiated the force of contraction during the final minute of the 20-Hz stimulation (P<0.05) with no effect of habituation. There was no effect of caffeine on 40-Hz stimulation strength nor was there an effect on maximal voluntary contraction or peak twitch torque. These data support the hypothesis that some of the ergogenic effect of caffeine in endurance exercise performance occurs directly at the skeletal muscle level.     

Influence of ryanodine receptor agonist and antagonist on development of potassium contracture in phasic (twitch) and tonic fibers of frog skeletal muscle

We compared the influence of external calcium and the inhibitor (dantrolene) and activator (4-chloro-m-cresol) of ryanodine-sensitive Ca channels of the sarcoplasmic reticulum on the characteristics of potassium contracture in phasic and tonic frog skeletal muscle fibers. The duration of contracture in tonic fibers, as contrasted to the phasic ones, is not limited by the presence of Ca²⁺. The tonic contractile response is virtually indifferent to dantrolene and is much less sensitive to chlorocresol than the phasic one (1 mM vs. 0.25 mM). In phasic fibers, the K⁺ contracture on the chlorocresol background is quite similar in amplitude and dynamics to that in control, whereas tonic fibers exhibit response summation without relaxation upon removal of excessive K⁺. One can suggest that in phasic fibers the Ca²⁺ influx can directly create a level sufficient to sustain contraction, while in tonic fibers its effect is mediated by Ca-dependent activation of the beta isoform of the ryanodine-sensitive channel.     

Calcium-induced calcium release mechanism from the sarcoplasmic reticulum in skinned crab muscle fibres

Mechanically skinned skeletal muscle fibres of the crab Carcinus maenas have been used to investigate the mechanism of calcium release from the sarcoplasmic reticulum. Calcium release has been monitored by the amplitude and kinetics of the tension developed by the fibre. Results show that a very low calcium concentration, insufficient to directly activate contractile proteins, induces a release of calcium from the SR. This release is stimulated by low concentrations of caffeine and inhibited by small amounts of EGTA. Thus, a graded calcium-induced calcium release mechanism dependent on extrareticular calcium concentration has been demonstrated in skinned crab muscle fibre.     

Water Loss during Contracture of Muscle

The relationship of contracture and exudation of water in frozenthawed frog muscle was studied. With maximum shortening, there was a water loss of 35 per cent of the weight of muscle. By restricting the contraction, it was demonstrated that the amount of water loss was proportional to the degree of shortening, there being no significant loss with isometric contraction. Muscle already shortened by tetanic stimulation also exuded water on subsequent freezing and thawing. The force of contraction could be reduced by depleting the muscle of calcium and it was shown that the amount of water exuded was also proportional to the tensile ability of the muscle. In a smooth muscle (anterior byssus retractor of Mytilus) which did not contract vigorously only a little water exuded. Contracture produced by caffeine was similarly associated with a loss of water. Microscopic studies revealed a disruption of the sarcomeres of the frozen-thawed muscle which contracted; glycerol-extracted and calcium-depleted muscles, which did not contract on freeze-thawing, did not show such disruption. Freezing and thawing of actomyosin caused a reversible syneresis of the protein. It is concluded that the exudation of the water is not merely due to the freezing and thawing but is also dependent on the contractile events.     

Effects of halothane, caffeine, dantrolene and tetracaine on the calcium permeability of skeletal sarcoplasmic reticulum of malignant hyperthermic pigs

Preparing skeletal sarcoplasmic reticulum from both normal and malignant hyperthermia susceptible pigs, the effects of various drugs on the passive calcium permeability of these sarcoplasmic reticulum preparations were studied. It was found that, in the absence of halothane, the permeability of heavy sarcoplasmic reticulum prepared from malignant hyperthermia susceptible pigs was much higher than that of normal pigs. It was observed that halothane, at concentrations above 10 microM (well below anesthetic concentrations, which are on the order of 1 mM), increased the permeability of sarcoplasmic reticulum. The Hill coefficient of the effect of halothane ranged from 1.96 to 2.25, suggesting that some kind of cooperativity was involved in this reaction. The effects of caffeine were similar to those of halothane. Inhibitors, such as tetracaine and ruthenium red inhibited both the calcium permeability and the halothane-induced increment. The Hill coefficient of the effect of tetracaine was 1.75. The mode of inhibition suggests that tetracaine directly binds with the calcium channel to inhibit the calcium efflux. On the contrary, dantrolene did not affect the calcium permeability of the sarcoplasmic reticulum. However, it inhibited the halothane-induced and caffeine-induced increments of the permeability. The Hill coefficient of inhibition by dantrolene ranged from 2.3 to 3.9, suggesting that several molecules of dantrolene may interact cooperatively with one calcium release channel to inhibit the effect of halothane. These results suggest that dantrolene has a unique inhibitory action, which may be related to its efficacy in ameliorating the syndrome of malignant hyperthermia.